196 Tubercle, Lond.

, (1970), 51, 196

OCCASIONAL SURVEY

THE HISTORY OF THE ZIEHL-NEELSEN STAIN

By P. J. BISHOP
from the Library, Institute of Diseases of' the Chest, London

and
G. NEUMANN
Stsidr, Gesundheitsamt,Abt. Tuberkulosefiirsorge, 7 Stuttgart 1, Schickhardtstrasse, 35, Germany

Although there are some good accounts in the literature of the history of staining the tubercle
bacillus, in particular those by the German historian of tuberculosis Predijhl (PredShl, 1888) and
Calmette (Calmette, 1923), many others are incomplete, contradictory, or even inaccurate. For
example William Bulloch in his history of bacteriology wrote-‘The so-called Ziehl-Neelsen
method was in reality Neelsen’s’ (p. 385) (Bulloch, 1938). This paper merely tries to trace the
chronology of events leading up the recognition of what is now known as the ‘Ziehl-Neelsen
method’, while at the same time reference is made to a few important or interesting early references
to the tubercle bacillus and its staining in some British journals of the period 1882-1887.
First of all it is necessary to say something about the two men eponymously commemorated in
this way. Friedrich Ziehl was born on the 13th April, 1857 at Wismar in Mecklenburg-Schwerin,
the son of a civil servant who later became an advocate. He studied and qualified at Heidelberg
in 1881. From 1882 until 1886 he was assistant in the Medical Clinic at Heidelberg, but he had to
leave for economii: reasons (the death of his father). From 1887 until his death on the 7th of April,
1926, he worked as a neurologist at Ltibeck, where he published several papers on neurological
subjects. The above details are taken partly from Fischer (Fischer, 1933) and partly from an
obituary published in a local Ltibeck journal. Fischer’s account would seem to be the only readily
accessible one in the literature. It stated that Ziehl’s name is known through his stain and gives the
reference to his 1882 paper. The Ltibeck obituary makes it clear that Ziehl was well-known in his
home town as the first to stain the tubercle bacillus with carbol fuchsin (Ltibeckische Blatter,
1926). No obituary of Ziehl appeared in the medical press, and as far as we have been able to find
out the only time he has been commemorated in recent years was in the paper by the late Dr. Walter
R. Bett (Bett, 1957).
Friedrich Karl Adolph Neelsen was born on the 29th March, 1854, at Uetersen, Holstein, the
son of a clergyman. He studied and qualified at Leipzig in 1876, where he was a pupil of Ernest
Wagner. From 1876 until 1878 he was an assistant at the Pathological Institute at Leipzig, and from
1878 until 1885 he occupied a similar position at Restock, where in 1884 he was made professor.
In 1885 he moved to Dresden, where he followed Birch-Hirschfeld as prosektor at the Stadtischem
Krankenhaus. He died on the 1lth of April, 1894. The above is again taken from Fischer (Fischer,
1933) who stated that he is remembered for the Ziehl-Neelsen stain, and also referred to his books:
his ‘Grundriss der pathologisch-anatomischen Technik’, Stuttgart, 1892, and a ‘Beitriige zur Pathologie’,
Vienna, 1888, written with F. M. Oberlander. Pagel’s account differs little from that of Hirsch
(Pagel, 1901). He gave a list of Neelsen’s writing, but did not mention his stain. He wrote ‘Neelsen
war ein ttichtiger Anathom und Patholog’. Apart from the above-mentioned accounts we have
been able to find only one informative obituary notice of Neelsen. This was written by a Dr. Grenser
and tells us that Neelsen published papers on disposition to disease in general; syphilis; carcinoma;
the role of bacteria within the body; methods of staining bacteria; spontaneous rupture of the
 ZIEHL-NEELSEN STAIN 197

heart; haemorrhagic infarction of the heart; and myocarditis (Grenser, 1893-94). It seems certain
that the cause of Neelsen’s death was tuberculosis. The symptoms appeared in the autumn of 1893,
and from the end of the year he suffered from emaciation, hoarseness, cough, and a general
deterioration. In the winter of 1893/94 he had intended to speak about carcinoma of the lung
(Dresden was the centre of cigarette manufacturing, and the increase in carcinoma of the lung was
first observed in Dresden), but he could not do this because of progressive hoarseness. He died on
the 1 lth of April, 1894, leaving a wife and a three year old son. Virchows Arc&v contained a note of
Neelsen’s death in a calendar of obituaries published at the end of the year, but it does not tell
us anything new. It gives as sources of information: Hirsch; Leopoldina; and Vossische Zeitung
Berlin. The first of these has already been mentioned. The Leopoldina reference merely records
Neelsen’s death. The third reference is to a newspaper, which we have been unable to see, but
which we strongly suspect only records Neelsen’s death.

KOCH’S DISCOVERYOF THE TUBERCLE BACILLUS

Koch’s formal announcement of his discovery of the tubercle bacillus was made on the 24th
of March, 1882. His first publication on the subject appeared in the Berliner Klinische Wochen-
schrif of April lOth, and bore the title-‘Die Aetiologie der Tuberculose’ (Koch, 1882). This
first paper included an account of the method he had used to stain the bacillus-
‘The material to be examined is prepared in the usual manner for examining for pathogenic
bacteria, and either spread on the cover slip, dried and heated or cut into sections after
fixation in alcohol. The cover slips or sections are placed in a staining solution of the
following constitution: 200~~. of distilled water are mixed with ICC. of a concentrated
alcoholic solution of methylene blue, shaken up, and then 0.2~~. of a 10% solution of
potassium hydroxide is added with repeated shaking. This mixture must show no precipitate
after standing for several days. The materials to be stained remain in this solution for
20 to 24 hours. By heating the solution to 40°C. in a water bath this time can be shortened
to a half to one hour. Following this the cover slips are covered with a concentrated aqueous
solution of vesuvin (Bismarck brown) which is filtered each time before using, and after
one to two minutes rinsed with distilled water. When the cover slips come from the methylene
blue, the attached layer appears dark blue and is markedly (p. 222) overstained. During the
treatment with vesuvin this blue colour is lost and it appears stained a faint brown. Under
the microscope all the constituents of animal tissue, that is, the cell nuclei and their products
of disintegration appear brown, while the tubercle bacilli, on the other hand, stain a
beautiful blue. Moreover, all other bacteria which I have investigated to date, with the
exception of the lepra bacilli, take on a brown color with this staining method. The color
contrast between the brown stained tissue and the blue tubercle bacilli is so striking that
the latter, which are present often only in very small number, nevertheless, are to be found
and identified with the greatest certainty’ (Pp. 227-28 in the original) (Koch, 1882) (the
translation is that of William de Rouville, 1938, Pp. 854-55.).
It is interesting to note that the three important factors in staining the tubercle bacillus: i.e. the
use of mordant, heat, and decolorizing all but the mycobacteria, are all included in Koch’s method,
recorded in his first paper of 1882. The present Ziehl-Neelsen stain has evolved from attempts to
improve on Koch’s method. The significant contributions to this would appear to be those of:-
Ehrlich (1882) who improved it by decolorization with a mineral acid-he also used a red
stain, fuchsin, and changed the mordant to aniline oil.
Ziehl(l882) who changed the mordant to carbolic acid.
Rindfleisch (1882) who heated the slide instead of putting it into hot water.
Neelsen (1883) who combined Ziehl’s mordant with Ehrlich’s red stain.
It is also interesting to relate these developments to others occurring during the same period,
and to examine chronologically some of the post-Koch (1882) literature.
198 BISHOP AND NEUMANN

EHRLICH
As we have said, the first suggestions for the improvement of Koch’s method came from Paul
Ehrlich, who was one of those present at Koch’s sensational announcement in Berlin. Ehrlich’s
communication was made to the Berlin Society of Internal Medicine on May lst, 1882. He recom-
mended staining the bacillus with aniline methyl violet instead of alkaline methylene blue:-
‘After these remarks 1 wish to briefly report the method. I have worked almost exclusively
with dry preparations, i.e. of sputa, but I have done controlled trials which have proved
that the method is also applicable to section preparations. I proceed by taking out a small
particle with a dissecting needle from the sputum and squeezing it flat between two cover-
glasses, and I have found that those of 0.10 to 0*12mm. are best. Under these conditions
it is easy to obtain uniformly thin layers from the small plug of sputum. Then both glasses
are torn from each other and one then gets two thin layers, which can easily be air-dried.
These preparations are not yet suitable. It is necessary to fix the protein. Usually I have done
this by keeping the preparations for one hour at 100 to 110°C. ; however there exists a more
convenient method which I have seen used in the Kaiserlichen Gesundheitsamt and which
consists in passing the air-dried preparations three times through the flame of a Bunsen
burner. For staining I use water, saturated by aniline oil, which may be prepared in a
few minutes by shaking water with surplus aniline oil and filtering it through a moistened
filter. To the water-clear fluid obtained in this way one adds drop by drop a saturated
alcoholic solution of fuchsin or methylene violet until a distinct opalescence indicates
saturation with the dye-stuff. Upon this fluid the preparations are made to float and staining
is adequate within a quarter to half and hour. The colour of the tubercle bacilli does not
disappear under the influence of vesuvin (Bismarck brown) or only very slowly. It is neces-
sary to use an acid and I soon turned to the use of strong, I may even say heroic, ones.
A mixture consisting of 1 volume of official nitric acid and 2 volumes of water can be
recommended. Under its influence one sees the preparation grow pale within a few seconds,
yellow clouds rise and the preparation becomes white.
If one examines the preparation at this stage, it is seen to be decolorized and only the
bacillus has maintained its intensive staining. It might be possible to examine such a
preparation, but the technical difficulties of focussing the bacillus are extremely great.
It is recommended to stain the background yellow if the bacilli are violet, and blue if they
are red.
I have succeeded in staining the bacillus with all basic aniline dyes, even with Bismarck
brown, and hence it follows that the substance of the bacillus itself does not differ from that
of other bacilli in its staining properties. But the discrimination of the tubercle bacillus
by staining depends on the presence of a surface layer with characteristic and specific
properties. The first of these to which Koch’s technique is pointing is that the covering
layer is permeable only to dyes under the influence of alkalis’ (Pp. 269-70) (Ehrlich, 1882).
One of the earliest references in the British literature can be found in an editorial in the Bri&
Medical Journal of May 13th, 1882. It begins :-
‘Experiments which have been carried on during the last few years have demonstrated
beyond a doubt that tuberculosis belongs to the group of infective diseases, although
various questions have arisen as to the origin of a similar disease from inoculation with
non-tubercular matter. Many of these difficulties have been cleared up, and a great advance
has been made in the pathology of this subject, by the beautiful experiments recently
published by Dr. Koch’ (Editorial, 1882).
This editorial includes a description of Koch’s method of staining. The same issue of the journal
refers to a meeting at the Physiological Laboratory at King’s College, London, on May 8th at
which Mr. Watson CheYne and Mr. E. M. Nelson had exhibited some specimens of the tuber&
bacillus, prepared by Koch, and brought over to London by a Dr. Goltdammer (British Medical
 ZIEHL-NEELSEN STAIN 199

Journal, 1882a). The British Medical Journal of June 17th, 1882, has a note entitled-‘Ehrlich’s
method of staining tuberele bacilli’ and we read:-
‘This method is a great improvement over Koch’s original method, Ehrlich has employed
it chiefly in the examination of sputa; and it has now been adopted by Dr. Koch, in
preference to his own method’ (British Medical Journal, 1882b).
Professor P. Baumgarten published a paper on June 24th, 1882, in which he described a staining
method using potash lye (Baumgarten, 1882). The British Medical Journal of July 22nd, 1882,
published a note under the heading ‘The bacillus of tubercle’:-
‘In reply to many questions which have been addressed to us on this subject, we may say
the bacilli of tubercle discovered by Dr. Koch are observed with difficulty, if his method of

FIG. 1
Franz Ziehl (1857-1926). A photograph taken between 1904 and 1908.

preparation be adopted. Dr. Ehrlich has devised a mode of preparation (published in this
Journal, June 17th, 1882), recently modified by Professor Rindfleisch of Wiirzburg, by
which their presence is more easily detected than by any other hitherto made known’
(British Medical Journal, 1882~).

ZIEHL
It is now that Franz Ziehl appears on the scene (Fig. 1). In his paper published on August 12th,
1882, he described how he had modified Ehrlich’s procedure by using carbolic acid instead of
aniline; and also by using a weaker acid for decolorization. The significant part of his paper would
seem to be:-
‘Up to the present we have not been able to obtain the pure constituents of aniline oil.
As it was necessary to use a substance belonging to the aromatic series we tested such a one,
the reaction of which was known to us from that second aniline oil, namely carbolic acid’
(p.451) (Ziehl, 1882).
The British Medical Journal of October 14th, 1882, carried two papers about staining the bacillus.
200 BISHOP AND NEUMANN

The first was by G. A. Heron, from the City of London Hospital for Diseases of the Chest, and
described in detail Ehrlich’s method. This was an eminently practical paper with special reference
to the author’s own experience (Heron, 1882). The second paper was by Heneage Gibbes, Curator
of the Anatomical Museum at King’s College. This paper was entitled-‘A new method for the
detection of the tubercle bacillus’. The author wrote that :-
‘Koch’s original method has utterly failed in the many cases in which I have tried it’
(Gibbes, 1882).
He also criticised Ehrlich’s modification, and suggested using two staining fluids: one, magenta,
to stain the bacillus; the other, chrysoidin, which stains the surrounding substance, but not the
bacillus.
The British Medical Journal of October 28th, 1882, published a paper by a William Vignal,
College de France, with the title-‘Some remarks on Ehrlich and Gibbes’s methods for the detection
of bacillus tuberculosis’ (Vignal, 1882). This paper is an indication of the interest that the subject
had already aroused. In the same journal of December 16th, 1882, we find a report of a meeting of
the Aberdeen, Banff, and Kincardine Branch, on November 22nd, at which Professor Alexander
Ogston gave a demonstration of the tubercle bacillus. Dr. Mackenzie Davidson, who had seen
the slides prepared by Dr. Heron of London, described the process. The remarks of a Dr. Simpson
(Medical Officer of Health) are of interest, as they reveal something of how some people still
thought :-
‘Dr. Simpson . . . questioned the connection between these bacilli and the diseases with
which they were associated. To him we seemed to be looking into a new world yet unknown,
and our connecting bacilli and disease as cause and effect seemed to have come too soon.
Bacilli were found everywhere, and it was a question unsettled in his mind whether or not
they really had any connection with disease’ (Simpson, 1882).
Dr. Schill’s paper of January lOth, 1883, on the identification of tubercle bacilli in sputum,
described Ehrlich’s method with Rindfleisch’s modification of heating the slide. Mention was also
made of Ziehl’s modification (Schill, 1883). Early in 1883, L. Lichtheim, of Berne, published a
paper which summarized the importance of finding tubercle bacilli in the sputum and in the faeces
(Lichtheim 1883). Ziehl’s second paper appeared on January 31st, 1883, and bore the title-‘The
importance of finding tubercle bacilli especially with regard to diagnosis and prognosis’. In this
paper Ziehl reported that he had found the bacilli in 73 cases of pulmonary tuberculosis, and he
finished his paper with the following conclusions:-
(1) In most cases of pulmonary tuberculosis tubercle bacilli may be found; exceptions do
occur.
(2) The finding of tubercle bacilli may confirm the diagnosis of tuberculosis. In some cases
the differential diagnosis is possible by this finding.
(3) If tubercle bacilli cannot be found, it is not always possible to exclude tuberculosis.
(4) The number and kind of tubercle bacilli do not allow any prognostic conclusions (Ziehl,
1883a).
The Lancet of February 24th, 1883, referred to a description by Professor Rosenstein, of Leyden,
of a case in which the bacilli were found in the urine in considerable numbers, and went on:-
‘The observations is of much interest, but at the same time rather by way of suggestion
than of proof. The question of the diagnostic value of tubercle bacilli is still in the region of
probable rather than of certain knowledge’ (Lancet, 1883a).
De Giacomi, of Berne, published a paper in March, 1883, in which he differentiated between
tubercle bacilli and other bacilli in the faeces (De Giacomi, 1883). The Lancet of March 17th, 1883,
had another note about tubercle bacilli in urine, and referred to a Dr. Babesiu of Pesth, who had
followed in parts the methods of Ehrlich and Koch, but instead of nitric acid had used strong acetic
acid (Lancet, 1883b). Ehrlich took part in a discussion of Koch’s discovery published in the
Deutsche Medizinische Wochenschrift of March 14th, 1883. He criticized a technique described by
 ZIEHL-NEELSEN STAIN 201

Spina as giving unfavourable results (Ehrlich, 1883). The Lancet under the heading ‘The tubercle
controversy’, referred in its issue of April 21st, 1883, to Dr. Spina’s monograph in which he sought
to minimize the importance of Koch’s discovery, and also mentioned the Vienna Allgemeine
Medizinische Zeitung, which apparently published a series of articles or reports under the heading-
‘The tubercle-bacillus war’ (Lancet, 1883~).
Ziehl published another paper on April 25th, 1883, entitled ‘On the staining of tubercle bacilli’.
This dealt with the acid-fastness of the bacilli, and his controversy with Ehrlich (Ziehl, 1883b).
Shortly afterwards, Heneage Gibbes, whom we have already mentioned, published a paper in the
Lancet of May 5th, 1883, in which he described-‘A rapid method of demonstrating the tubercle

FIG. 2
Friedrich Carl Adolf Neelsen (1854-94). A photograph taken between 1885 and 1894.

bacillus without the use of nitric acid’ (Gibbes, 1883). Carl Weigert, who was a cousin of Ehrlich
published on June 13th, 1883, a paper-‘New notes concerning the pathogenesis of acute generalized
miliary tuberculosis’. In this he stated that he had given up using aniline oil:-
‘As it is not always possible to rely upon the aniline oil, as Ziehl has found, I have given
up its use completely, and I make use of a 2% filtered aqueous solution of gentian violet to
which I add half per cent liquor ammonii caustici (It is possible to raise the concentration
up to l$%. It is known from the experience of potash dyers that lye is harmful to methyl
violet). This solution becomes stable for weeks by adding 10% alcohol, but it may be better
to prepare it new every time. This is simple even without special laboratory equipment if
one has a pipette graded in cubic centimeters and a graduated cylinder. It is best to stain
for 20 to 30 minutes at incubator temperature, or over a longer time at room temperature,
and one then continues as described by Koch and Ehrlich’ (p. 351) (Weigert, 1883).

NEELSEN

Friedrich Neelsen now comes into the story (Fig. 2). His first publication on the subject would
appear to have been in the German abstracting journal-Centralblatt ,fiir die medicinischen
202 BISHOP AND NEUMANN

Wissenschuften-of July 14th, 1883, which at that time published a considerable number of original
papers. Neelsen’s article had the rather general title-‘A casuistic note on the theory of tuberculosis’,
but included a footnote :-
‘In this case I used first as a staining fluid a three-quarter per cent solution of fuchsin in
5 % carbolic acid with admixture of some alcohol and decolorised with 25 % sulphuric acid.
This method, which I have used almost exclusively for a long time, gives in my hands
better results than the other methods (perhaps only because I am most accustomed to it)’
(p. 500 (Neelsen, 1883).
Prior, of Bonn, published a paper on staining the bacillus on August 13th, 1883, which discussed
the acid fastness (Prior, 1883). The Lancet of November lOth, 1883, reported that:-
‘Opinion is still divided in Vienna as to the significance of the bacillus tuberculosis, but
the record of the last meeting of the Medical Society there shows that not every observer has
arrived at the same negative conclusions as Spina. For on that occasion a paper was read
by Dr. Heitler on the diagnostic and prognostic importance of the tubercle bacillus, in
which he maintained that those only could claim to speak with authority who had repeated
with every precaution the experiments of Koch’ (Lancet, 1883d).
On November 26th, 1883, Dr. Petri, writing from Dr. Brehmer’s sanatorium at Gijrbersdorf,
referred to staining by fuchsin and malachite green, using glacial acetic acid (Petri, 1883). In 1884
Koch published his important, second article, or rather monograph on ‘Die Aetiologie der
Tuberculose’ (Koch, 1884). In the words of Dr. Arthur L. Bloomfield:-
‘In this important article of eighty-six folio pages Koch makes his definitive presentation
of the discovery and isolation of the tubercle bacillus and the proof of its causal relation
to the disease. It is really a masterful elaboration of his preliminary communication’
(Bloomfield, 1954).
Copies of the Berliner Klinische Wochenschrift of 1882 and the ‘Mittheilungen aus dem Kaiserlichen
Gesundheitsamte’ of 1884 are to be found only in the larger, older medical libraries, so we have
indicated in our bibliography those more accessible sources where readers may find them reprinted.
Dr. B. Frankel’s lecture to the Berlin Medical Society on. . ‘The staining of Koch’s bacillus and its
semiotic importance for diseases of the respiratory tract’ was published on April 7th, 1884. It
contained a description of the laryngeal swab technique by direct touch, and notes on the prognostic
value of bacilli in sputum (Frankel, 1884). A Lancet editorial of March 15th, 1884, referred to
Koch’s 1884 work as :-
‘The very lucid and exhaustive monograph upon the etiology of tuberculosis by Dr. R.
Koch, which, although it has been written for some months, has only just now been given
to the world, serves to place forcibly before the profession the pathological facts upon which
the doctrine of the infectivity of tubercle, and the subsidiary, yet more important, doctrine
of the contagiousness of phthisis, are based’ (Lancet, 1884).
The above editorial is very interesting and informative, but space does not allow us to quote
more of it.
The Lancet of January 17th, 1885, contains an extremely interesting and illuminating account of a
discussion on the tubercle bacillus and the pathology of phthisis held at the Royal Medical and
Chirurgical Society on January 13th. Specimens of the bacillus, prepared by Mr. Watson Cheyne
and Dr. Percy Kidd were on view. One of the speakers was Dr. Green. This would almost certainly
have been T. Henry Green of the Brompton Hospital-a noted pathologist. Referring to scientific
criticism of Koch’s discovery ‘he considered that the question was one for experimental pathologists
to investigate, and so far experimental pathology confirmed Koch’s and Watson Cheyne’s
observations’ (Lancet, 1885). Another speaker was Wilson Fox, one of the greatest authorities of his
day on tuberculosis. He is reported as saying that:-
‘Although the evidence in favour of Koch’s views was so strong, yet, looking to the
constant change in our views of phthisis, and seeing how the pathology of tuberculosis
 ZIEHL-NEELSEN STAIN 203

has biennially, triennially, or quinquennially been swayed by histological dogmata, it would

be well if we waited before definitely accepting all that the new views implied. Who could tell
but that in a few years we should hear expressions in terms which at present we cannot
foreshadow? It should be remembered that the bacteridia of tubercle depended for their
detection on one of the most uncertain of chemical agents, an aniline dye’ (Fox, 1885).
Ehrlich’s paper ‘The biological utilisation by methylene blue’ was published on February 21st,
1885, but he had in fact introduced this as early as 1881 (Ehrlich, 1881; 1885). In 1885 Professor
Heinrich Albert Johne (1839-1910) remembered for his discovery of Johne’s bacillus, and
description of Johne’s disease, and also for a pioneer German history of tuberculosis, published a
paper ‘An undoubted case of congenital tuberculosis’. In an interesting footnote he makes mention
of Neelsen, and thereby brings him into the story again:-
‘This previously unpublished method is one of the modifications of the Ehrlich-Weigert-
Koch method described by Ziehl (Deutsche Med. Wochenschr. 1882, No. 33, u 1883, No. 17)
by which aniline oil is replaced by carbolic acid. Its preparation and application, for which
I am indebted to a courteous personal communication of Herrn Neelsen, is as follows:
1,0 fuchsin is dissolved in 1OOg. of an aqueous 5% solution of carbolic acid and to this
solution is added log. of alcohol. Staining of the cover-glass preparation with heat up to the
rising of steam is very quick and intensive. In microtome sections the bacilli are surprisingly
well stained at room temperature within 5 to 10 minutes. Decolorize with a 25% aqueous
solution of sulphuric acid, counterstaining with methylene blue. The solution can be stored
for several weeks without any change in its excellent staining properties’ (footnote 1,
Pp. 200-01) (Johne, 1885).
Ehrlich published a long paper in 1886 in which he reported on his studies with aniline and its
derivatives, the importance of the waxy envelope of tubercle bacilli, the role of counterstaining,
and the importance of repeating the staining of the bacilli (Ehrlich, 1886).

ZIEHL-NEELSEN

The Lancet of April 9th, 1887, published a letter from a Dr. Henry S. Gabbett, of Eastbourne,
entitled ‘Rapid staining of the tubercle bacillus’. This is of interest as it refers to Neelsen’s method
by name. Gabbett advocated using a stain made from one part of magenta in 100 of a 5 % watery
solution of carbolic acid, and adding 10 of absolute alcohol. He considered this prefereable to any
other stains containing solutions of aniline (Gabbett, 1887). In 1892, only two years before his
early death, Friedrich Neelsen published a little book-essentials of pathological-anatomical
technique for practitioners and students. In it he described his method of staining:-
‘For staining tubercle bacilli on the coverglass I use the ordinary solution of carbolic
acid and fuchsin (1.0 fuchsin, 100-O 5% aqueous solution of carbolic acid, 10.0 absolute
alcohol) in the following way. The dried cover-glass preparations, heated for 5 minutes to
llO”C., are covered by means of a pipette with one large drop of the staining fluid and
placed on the copper sheet which serves for heating. (One has to be careful that no fluid
runs off the cover-glass, spraying about, and often disturbing the cover-glass). As soon as
the fluid has come to the boil which only takes a few seconds, a small drop of staining
solution is added to avoid dehydration, and this is repeated so that the preparations remain
covered with boiling stain solution for 14 to 2 minutes (measured by a watch). After this
they are put into an aqueous solution of sulphuric acid (25.0 to 75-O distilled water) where
they left for half to two minutes until the dark-red colour has almost completely disappeared.
During the succeeding washing with abundant (ordinary) water the red colour is partly
restored. If the preparations still appear as dark-red, decolorizing with sulphuric acid
must be repeated until they are only a pale red colour. Finally the cover glasses are dipped
for counterstaining for half to one minute into a concentrated aqueous solution of methylene

G
204 BISHOP AND NEUMANN

blue, again rinsed in water, dried, and mounted in balsam. The tubercle bacilli appear in the
preparation bright red, the nuclei blue, and the cytoplasm pale blue’ (Pp. 66-67) (Neelsen,
1892).
This description undoubtedly corresponds to the so-called Ziehl-Neelsen technique of staining
tubercle bacilli as used up to now.
In 1888 Predohl published his history of tuberculosis; one of those invaluable early works to
which most later historians are indebted. In it he reported on all the methods of staining described
between 1882 and 1888. The modification of Ziehl is mentioned as well as that of Neelsen, but a
literal translation of his wording would be-‘1 must describe the modification of Ziehl’s method
given by Neelsen’ (Predohl, 1888, p. 371). In other words, the Ziehl-Neelsen method is not actually
referred to in those terms.
The phrase ‘Ziehl-Neelsen’ technique (or method) was not in use before the early 1890’s. In
1893 in America, Surgeon-General George M. Sternberg (1838-1915), said to have been the first
man in the United States to photograph the tubercle bacillus, published a manual of bacteriology-
one of the first works in English on the subject. His book included a list and details of the various
methods of staining the tubercle bacillus known at that time, and he actually listed the Ziehl-Neelsen
method by name (Sternberg, 1893).
One of the first, if not the first, manuals of bacteriology published in England was written by
Edgar March Crookshank (1858-1928), a pupil of Koch who later became Professor of Bacteriology
at King’s College, London. His first edition of 1886 contained a good list of methods of staining
with details, and although his list included both Ziehl’s and Neelsen’s methods, no mention was
made of the Ziehl-Neelsen method as such. His second edition of 1887 and his third edition of 1890
made no change as far as this was concerned; but in his fourth edition of 1896 he wrote that of the
numerous methods of examining the tubercle bacillus few will be described as many are only of
historical interest. He did, however, say that-‘The Ziehl-Neelsen method is preferred by the author
for sections and cover-glass preparations’ (Pp. 384-87). (Crookshank, 1896).
It would appear then that sometime between 1890 and 1893, the Ziehl-Neelsen method had
become known and labelled as such.
In reality the Ziehl-Neelsen technique is based on the work of at least five people: Koch, Ehrlich,
Rindfleisch, Ziehl, and Neelsen. So the correct name should be : Koch-Ehrlich-Rindfleisch-Ziehl-
Neelsen method, a name which stands little chance of being used in practice. In some ways it
seems justified to continue using the well-known name ‘Ziehl-Neelsen method’. The publications
of these two workers were the last in which an important modification of the original method
was recommended, modifications which are still in use. Neelsen’s description in particular, is as
complete as it is correct, and furthermore is still not obsolete. The participation of other workers
means that we cannot be quite satisfied with this eponym. But in reality many such names exist in
medical terminology, well established and almost impossible to eradicate; and many are more
incorrect than the name for this method of staining tubercle bacilli. This cannot, and indeed should
not serve as a source of consolation, but rather as an explanation.
Ziehl died in 1926 and Neelsen in 1894, the latter at the relatively early age of forty.
Now, more than eighty years after the first description of the technique, the names of these two
German doctors are known all over the world wherever the tubercle bacillus is sought.
We are much indebted to Dr. E. Ziehl, of Liibeck, for letting us have the photograph of his father, and to Dr. P.
Scheid, Direktor des Pathologischen Institutes Dresden-Friedrichstadt, for the photograph of Neelsen, one of his
predecessors. Without their co-operation we should have been unable to include photographs of Ziehl and Neelsen.

REFERENCES
BAUMGARTEN,
P. (1882). Ueber ein bequemes Verfahren, Tuberkelbacillen in Sputis nachzuweisen. CenrralMrrr fiir
die medicinischen Wissenschaften, 20, 433.
BETT, W. R. (1957). Remembered solely for a stain: Franz Ziehl (1857-1926). N.A.P.T. (National Association forthe
Prevention of Tuberculosis) Bulletin, 20, 60.
 ZIEHL-NEELSEN STAIN 205

BLOOMFIELD,A. L. (1954). A bibliography of internal medicine: tuberculosis. Stanford Medical Bulletin, 12, 217.
BRITISHMEDICALJOURNAL(1882a). The bacilli of tubercle. British Medical Journal, i, 709.
BRITISHMEDICALJOURNAL(1882b). Ehrlich’s method of staining tubercle bacillus. British Medical Journal, i, 916.
BIUTISHMEDICALJOURNAL(1882~). The bacillus of tubercle. British Medical Journal, ii, 142.
BULLOCH,W. (1938). The History of Bacteriology, Pp. 216-17; & 385, Oxford University Press, London.
CALMETTE,A. (1923). Tubercle Bacillus Infection in Man and Animals, authorized translation by W. B. Soper and
G. H. Smith, Pp. 14-21, Williams & Wilkins, Baltimore.
CROOKSHANK, E. M. (1896). A Text-Book of Bacteriology, 4th edition, Pp. 384-87, H. K. Lewis, London (1st edition,
1886; 2nd edition 1887; 3rd edition, 1890).
DE GIACOMI,-. (1883). Dte diagnostische Bedeutung des Nachweises der Tuberkelbacillen im Stuhl. Fortschritte
der Medizin, 1, 145.
EDITORIAL(1882). Recent researches on bacteria. II. Koch’s investigation on tuberculosis. British Medical Journal,
i, 706.
EHRLICH. P. (1881). Ueber das Methylenblau und seine klinisch-bakterioskopische Verwerthung. Zeitschrift fiir
klinische Medizin, 2, 710.
EHRLICH,P. (1882). V. Aus dem Verein fur innere Medizin zu Berlin. Sitzung vom 1. Mai 1882. Deutsche Medizinische
Wochenschrift, 8, 269.
EHRLICH, P. (1883). Referat und Discussion tiber die gegen R. Koch’s Entdeckung der Tuberkelbacillen neuerlichst
hervorgetretenen Einwlnde. Deutsche Medizinische Wochenschrift, 9, 159.
EHRLICH, P. (1885). Zur biologischen Verwertung des Methylenblau. Centralblatt fiir die Medicinischen Wissen-
schaften, 23, 113.
EHRLICH, P. (1886). Beitrage zur Theorie der Bacillenflrbung. Chqrite-Annalen, 11, 123.
FISCHER, I. (1933). Biographisches Lexikon der hervorragenden Arzte der letzten ftinfzig Jahre, 2, 1104 & 1723,
Urban und Schwarzenberg, Berlin und Wien.
Fox, W. (1885). Royal Medical and Chirurgical Society. Discussion on the tubercle bacillus and on the pathology
of phthisis. Lancet, i, 107.
FRANKEL, B. (1884). III. Ueber die F&bung des Koch’schen Bacillus und seine semiotische Bedeutung fur die
Krankheiten der Respirationsorgane. Berliner klinische Wochenschrift, 21, 214.
GABBE’TT, H. S. (1887). Rapid staining of the tubercle bacillus. Lancet, i, 757.
GIBBES,H. (1882). A new method for the detection of the tubercle bacillus. British Medical Journal, ii, 735.
GIBBES,H. (1883). A rapid method of demonstrating the tubercle bacillus without the use of nitric acid. Lancet, i, 771.
GRENSER,-. (1893-94). XXI. Sitzung am 14. April, 1894. Jahresberichte der Gesellschaft fiir Natur- und Heilkunde
in Dresden, PP. 85-87.
HERON, G. A. (1882). Ehrlich’s method for the detection of tubercle-bacillus in sputum. British Medical Journal,
ii. 715.
--7 .--.

JOHNE, J. (1885). Ein zweifelloser Fall von congenitaler Tuberculose. Fortschritte der Medizin, 3, 198.
KOCH. R. (1882). Die Aetiolozie der Tuberkulose. Berliner klinische Wochenschrift. 19. 221.
KOCH; R. (1932). The Aetiology of Tuberculosis. A translation from the German of the original paper . . . specially
prepared by the American Review of Tuberculosis,, March, 1932, by Dr. and Mrs. Max Pinner. With an intro-
duction by Allen K. Krause. National Tuberculosts Assoctation, March, 1932, New York City.
KOCH, R. (1938). The etiology of tuberculosis. (English translation by William deRouville). Medical Classics, 2, 835.
(The above publication contains the original text of Koch’s 1882 paper together with an English translation, and
a bio-bibliography).
KOCH, R. (1953). Die Aetiologie der Tuberculose. Tuberkulose-Jahrbuch, 1951/52, p. 167. (A modern reprint of
Koch’s 1882 paper).
KOCH, R. (1884). Die Aetiologie der Tuberkulose. Mittheilungen aus dent Kaiserlichen Gesundheitsamt, 2, 1.
KOCH, R. (1886). The etiology of tuberculosis. In Recent Essays by various authors on Bacteria in Relation to Disease,
selected and edited by W. Watson Cheyne, New Sydenham Society, London. (The above contains an English
translation of Koch’s 1884 monograph-by Stanley Boyd, p. 67).
LANCET(1883a). Tubercle bacilli in urine. Lancer, i, 333.
LANCET(1883b). Tubercle bacilli in urine. Lancet, i, 464.
LANCET(1883~). The tubercle controversy. Lancet, i, 699.
LANCET(1883d).The tubercle bacillus. Lancet, ii, 827.
LANCET(1884). Editorial?-no title. Lancet, i, 480.
LEOPOLDINA(Leopoldina-Carolinischen Deutsche Akademie der Naturforscher) 1894, 30, 109.
LICHTHEIM.L. (1883). Zur diaanostischen Verwerthunc der Tuberkelbacillen. Fortschritte der Medizin. 1. 4.
L~~BECKISC~EBLb;TT&-Zeit.&hrift der Gesellschaft & Beforderung gemeinniitziger Ttitigkeit. Lube&, Sonntag,
25th April, 1926 (Nr. 17) (Dr. med. Franz Ziehlt).
NEELSEN, F. (1883). Ein casuistischer Beitrag zur Lehre von der Tuberkulose. Centralblatt fiir die medicinischen
Wissenschaften, No. 28, p. 497.
NEELSEN, F. (1892). Grundriss der pathologisch-anatomischen Technik fur praktische Arzte und Studierende, p. 66,
Ferdinand Enke, Stuttgart.
PAGEL, J. L. (1901). Biographisches Lexikon hervorragender Arzte des neunzehnten Jahrhunderts, Pp. 1192-93,
Urban und Schwarz enberg, Berlin und Wien.
PETRI, -. (1883). 11. Zur Farbung des Koch’schen Bacillus in sputis, sowie tiber das gleiche Verhalten einiger
Pilzzellen. Berliner klinische Wochenschrift, 20, 739.
PREDGHL,A. (1888). Die Geschichte der Tuberkulose, Pp. 356-71, Leopold Voss, Hamburg und Leipzig.
PRIOR, -. (1883). Beitrag zur Farbbarkeit des Tuberkelbacillus. Berliner klinische Wochenschrifl, 20, 497.
206 BISHOP AND NEUMANN

SCHILL, -. (1883). II. Ueber den Nachweis von Tuberkelbacillen im Sputum. Deutsche Medizinische Wochenschrift,
9, 15.
S~SON, -. (1882). Contribution to discussion. British Medical Journal, ii, 1213.
STERNBERG,G. M. (1893). A Manual of Bacteriology, p. 30, William Wood, New York.
VIGNAL, W. (1882). Some remarks on Ehrlich and Gibbes’s methods for the detection of bacillus tuberculosis.
British Medical Journal, ii, 826.
V~RCHOWSArchiv fur pathologische Anatomic und Physiologie und fiir die klinische Medizin, 1895, 139, 564.
WEIGERT,C. (1883). I. Neue Mittheihmgen uber die Pathogenie der acuten allgemeinen Miliartuberculose. Deutsche
Medizinische Wochenschrzft, 9, 349.
ZIEHL, F. (1882). Zur Farbung des Tuberkelbaclilus. Deutsche Medizinische Wochenschrif, 8,451.
ZIEHL, F. (1883a). Zur Lehre von den Tuberkelbacillen insbesondere tiber deren Bedeutung fur Diagnose und
Prognose. Deutsche Medizinische Wochenschrift, 9, 62.
ZIEHL, F. (1883b). Ueber die Farbung des Tuberkelbacillus. Deutsche Medizinische Wochenschr#. 9, 247.